A previously reported autofluorescence microspectroscopy protocol for fibroblasts25 was adopted for hMSCs based on their similar adherent nature. Selected passages of hMSCs were seeded at a concentration of at least 3.0 × 104 cells/ml on glass coverslips (Schott) within square silicone wells fabricated from medical grade silicone (Wacker Chemie AG). These coverslips with cells in 1 ml of StemMACS media (Miltenyi Biotec) were then incubated in a CO2 incubator for 24 h before autofluorescence microspectroscopy. Prior to measurements, the culture media was extracted and the remaining contents in the wells were washed twice with 1 ml of PBS. After washing, any excess PBS was remove and 100 μl of imaging solution (Thermo Fisher Scientific) was added. Five phase contrast images were obtained per coverslip at random locations through a 4× objective. Autofluorescence images and spectra were then taken through a 60× oil-immersion super apochromat objective (Olympus). At least 100 cells per early and senescent passage per donor were involved in each autofluorescence run. Autofluorescence output of at least 10 cells were recorded with measurements made at different locations within the silicon well (experimental repeats n = 10). This was followed by five background spectral measurements of a 100 μl volume of imaging solution placed on a clean region of the same coverslip.

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